Alkaline phosphatase (EC 3.1.3.1.) (abbreviation:ALP) is a metalloenzyme consisting of a group of isoenzymes present in different tissues of animal organisms and in particular in man.
Alkaline phosphatase isoenzymes are important in protocols for diagnosing different conditions in adults or in children, and a number of methods have been proposed for separating and assaying such isoenzymes. ALP isoenzymes can be divided into four classes: non-specific tissue (bone, liver and kidney), adult intestinal, foetal intestinal, and placental. A number of variations can exist within a single class, namely:
hepatic: hepatic 1 (H1), hepatic 2 (H2); PA1 ultrafast (UF); PA1 osseous (Os); PA1 placental: placental 1 (P1), placental 2 (P2); PA1 intestinal: intestinal 1 (I1), intestinal 2 (I2), intestinal 3 (I3). PA1 depositing the biological sample containing the ALP isoenzymes to be separated on the electrophoresis support; PA1 depositing a lectin solution which can interact with the ALP isoenzymes contained in the sample on the electrophoresis support; PA1 applying an electric field to permit electrophoretic separation by migration of the ALP isoenzymes under conditions which can permit differential separation of the osseous and hepatic ALP fractions; PA1 revealing the separated ALP isoenzymes. PA1 an electrophoresis support comprising a porous material suitable for depositing a biological sample to be analysed and for carrying out electrophoretic migration; PA1 a solution of lectin in a concentration in the range of 0.1 mg/ml to 15 mg/ml, advantageously in the range of 1 to 10 mg/ml, preferably in the range of 1 to 10 mg/ml.
Thus nine principal fractions can be distinguished which have to be separated, identified and quantified in particular for their detection into hepatic and biliary disorders and into certain bone diseases, including osseous tumours or Paget's disease.
Reference will occasionally be made below to the term "fraction" to designate a class of ALP isoenzymes or a particular variant within a class of isoenzymes. On the electrophoresis support, a "fraction" corresponds to a band revealed after migration.
The most frequent routine analysis carried out on alkaline phosphatase isoenzymes consists of measuring the total enzymatic activity using a substrate of this enzyme, generally para-nitrophenylphosphate. That method, however, cannot determine the levels of the different isoenzymes.
The principal method for separative analysis of such compounds uses electrophoretic techniques. Isoelectrofocussing is occasionally used and can separate 10 to 20 bands depending on the procedure used. Identifying all of the bands is difficult, rendering clinical interpretation extremely awkward.
Zone electrophoresis enables a good separation of the principal forms of the isophosphatases. However, certain fractions are superimposed, in particular the Os, H1 and P1 fractions, and thus complementary treatments have to be carried out to separate and identify them. Such treatments must be carried out on the biological test samples to be tested before depositing them onto the gel.
Such treatments consist, for example, of thermal denaturing, incubation with specific inhibitors such as urea, amino acids, etc., enzymatic incubation with neuraminidase, ficin, phospholipase C, incubation with specific antiplacental or anti-intestinal antisera.
Several separation procedures which are in current use have been dealt with by Van Hoof V. O., De Broe Marc, E., Clinical Laboratory Sciences, vol. 31, issue 3 1994, "Interpretation and clinical significance of alkaline phosphatase isoenzyme patterns".
One particular procedure has been proposed in U.S. Pat. No. 5,264,098 which describes the separation of ALP isoenzymes using a gel electrophoresis reaction employing a gel buffer containing at least one non ionic detergent and an anionic detergent.
Available treatments for identifying and quantifying ALP isoenzymes have certain disadvantages as regards routine analysis. In addition to high costs, they can on the one hand be long and can considerably complicate manipulation, and on the other hand, complete determination (of all of the isoenzymes) necessitates a plurality of treatments (2 or 3) for a single sample, limiting the number of samples which can be simultaneously analysed on the one gel.
Other treatments have been proposed which, for example, recommend treating the sample prior to loading onto the electrophoresis gel. In this regards, the action of the WGA lectin (wheat germ agglutinin) is particularly interesting (see Sidney B. Rosalki, A. Ying Foo, Clinical Chemistry, 30/7, p. 1182-1186, 1984, "Two methods for separating and quantifying bone and liver alkaline phosphatase isoenzyme in plasma", European patent EP-A-0 131 606 dated May 11, 1986). EP-A-0 131 606 describes the differential detection of bone and liver ALP isoenzyme comprising treating the test sample with lectin, then incubating the mixture obtained followed by separating the ALP bound to the lectin from the fraction containing free ALP and determining the ALP activity in one of the two media or in both. In a particular implementation of that patent, the two fractions (ALP bound to lectin and free ALP) are separated by electrophoresis.
With the exception of the intestinal forms, all isophosphatases possess sialic acids and are thus affected by a treatment with WGA lectin to a greater or lesser extent. The osseous fraction is the most sialated and thus is affected the most by this treatment, which under suitable conditions retards its mobility and thus causes it to precipitate in a zone which is distinct from the zone where the hepatic fraction is located.
In order to render ALP isoenzyme precipitation more selective towards the osseous isoenzyme, certain authors have used detergents such as Triton X100 (Rosalki). However, despite the presence of such detergents, residual interactions of the WGA lectin with other isophosphatases subsist, which cause co-precipitation of such fractions with the osseous fraction.
In addition to this lack of specificity, a further disadvantage of this technique is to render the analysis considerably more complicated.
The publication by Rosalki S. B. et al, cited above, alternatively proposes incorporating lectin into the buffer used to impregnate the electrophoresis gel prior to using this gel. This dispenses with prior treatment of the sample. In that case, the majority of the osseous fraction is precipitated close to where the sample has been loaded. The mobility of all of the other isoenzymes with the exception of intestinal isoenzymes is affected by the action of the WGA lectin despite the presence of the detergents mentioned above.
In the context of that treatment, the properties of the lectin used are its ability to interact specifically with the ALP isoenzymes which contain sialic acids.